Department of Cell Biology, UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UKMRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London WC1E 6BT, UK

ABSTRACT

At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in RasV12-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA–VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis.

Footnotes

K.A.A. performed the experiments and analyzed the results. K.A.A., K.M. and Y.F. conceived the experiments and wrote the manuscript. K.A.A., C.J. and Y.F. designed the SILAC screen. J.S. performed HILIC and LC-MS/MS. A.O., M.K., S.I. and M.B contributed to different aspects of experimental work and data interpretation. P.M.B. and T.R. contributed to VASP constructs. K.M. and Y.F. contributed equally to this work.

Funding

K.A.A. was supported by a fellowship from the Medial Research Council. T.R. received support from Vetenskapsrådet [grant number 21462]; the German Research Society [grant number SFB 841]; and the European Research Council [grant number ERC-StG-2012-311575_F-12]. C.J. is supported by a Cancer Research UK Career Establishment Award [grant number A12905]. K.M. and M.S.B. were supported by the The Wellcome Trust [grant numbers 084678/Z/08/Z and 099173/Z/12/Z]; and the Biotechnology and Biological Sciences Research Council. Y.F. is supported by Next Generation World-Leading Researchers (NEXT Program); the Takeda Science Foundation; the Uehara Memorial Foundation; Daiichi-Sankyo Foundation of Life Science; and Naito Foundation. Deposited in PMC for release after 6 months.

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